Methods of bead manipulation and forming bead arrays

ABSTRACT

According to various embodiments, a method is provided that comprises washing an array of DNA-coated beads on a substrate, with a wash solution to remove stacked beads from the substrate. The wash solution can include inert solid beads in a carrier. The DNA-coated beads can have an average diameter and the solid beads in the wash solution can have an average diameter that is at least twice the diameter of the DNA-coated beads. The washing can form dislodged DNA-coated beads and a monolayer of DNA-coated beads. In some embodiments, first beads for forming an array are contacted with a poly(ethylene glycol) (PEG) solution comprising a PEG having a molecular weight of about 350 Da or less. In some embodiments, slides for forming bead arrays are provided as are systems for imaging the same.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/872,333, filed on Aug. 31, 2010, which claims priority pursuant to 35U.S.C. §119(e) to U.S. Provisional Patent Application Ser. No.61/238,633, filed on Aug. 31, 2009, entitled “Enhanced Systems andMethods For Sequence Detection,” U.S. Provisional Patent ApplicationSer. No. 61/238,667, filed on Aug. 31, 2009, entitled “Enhanced Flowcelland Reagent Delivery For Sequence Detection,” U.S. Provisional PatentApplication Ser. No. 61/307,623, filed on Feb. 24, 2010, entitled“Methods of Bead Manipulation and Forming Bead Arrays,” U.S. ProvisionalPatent Application Ser. No. 61/307,492, filed on Feb. 24, 2010, entitled“Flowcells and Methods of Filling and Using Same,” U.S. ProvisionalPatent Application Ser. No. 61/307,641, filed on Feb. 24, 2010, entitled“Flowcells and Methods of Filling and Using Same,” and U.S. ProvisionalPatent Application Ser. No. 61/307,486, filed on Feb. 24, 2010, entitled“Flowcell, Flowcell Delivery System, Reagent Delivery System, and MethodFor Sequence Detection,” the entirety of each of these applicationsbeing incorporated herein by reference thereto.

FIELD

The present disclosure is directed towards the field of molecularsequencing, in particular towards of solid-support/bead handling andforming ordered-arrays.

BACKGROUND

There is a challenge to pack beads modified with nucleic acid molecules,closely together to form an array for sequencing. It would be desirableto place nucleic acid-coated beads in an organized, tightly packedfashion, for example, to increase throughput per cycle and to lowercustomer cost per sequenced base. As the bead deposition density isincreased, however, the likelihood of bead clumping and bead stackingincreases. Controlled organization of the bead would also simplifysoftware identification of the beads on the surface. Unfortunately, whenthe beads are stacked or clumped, there can be problems withinterrogation for their individual reporter signals.

In sequencing using beads, the overall throughput in terms of nucleicacid bases sequenced per sequencing run is directly dependent on thenumber of readable beads in a given interrogation area, and generally,the more the better. When beads are dispensed randomly onto a slide, aconsiderable amount of space on the slide is left open. Furthermore,some beads settle on the slide overlapping and/or stacking with severalother beads, which can cause difficulties in resolving and interpretingimages of the beads.

When imaging an array of beads, for example, fluorescently labeled beadsfor nucleic acid sequencing, it is desirable to have the beads packed asdensely as possible to achieve the highest throughput. However, issuesmay arise for beads of a size such that the diffraction circles orspread function is relatively large compared to the actual bead size.For example, a one micron diameter bead could have a diffraction circleof about 2 microns. Packing the beads at a density such that the beadsare all or mostly all touching each other results in un-resolvablefeatures, whether these beads are randomly arrayed or ordered in a closepack.

SUMMARY

According to various embodiments, a method is provided that includeswashing an array of first beads on a substrate with a wash solution toremove stacked beads from the substrate. The wash solution can comprisefrom about 2% by weight to about 50% by weight inert solid beads in acarrier. The solid beads in the wash solution can have an averagediameter that is larger than the first beads, for example, at leasttwice the diameter of the first beads. The washing can form dislodgedfirst beads. The method can comprise removing the wash solution and thedislodged first beads from the substrate to form a monolayer of firstbeads. In some embodiments, the substrate can comprise a slide. In someembodiments, the substrate can comprise a plurality of channels formedin a surface thereof. The first beads can comprise DNA-coated beads. Insome embodiments, the wash solution can comprise first abrasive beadshaving a second average diameter, and second abrasive beads having athird average diameter, and the second average diameter can be about 50%greater than the third average diameter.

According to various embodiments, a method of forming an ordered arrayof beads is provided that comprises contacting a plurality of firstbeads with a poly(ethylene glycol) (“PEG”) solution comprising a PEGhaving an average molecular weight of about 350 Da or less, to form abead mixture, and depositing the bead mixture on a surface of an arraysubstrate. In some embodiments, the PEG can have a molecular weight ofabout 200 Da. The array substrate can comprise a slide having aplurality of grooves formed in a surface thereof, and the bead mixturecan be deposited into the grooves.

According to various embodiments, an ordered array of beads is providedthat comprises a substrate comprising hydrophobic lines in the shape ofa grid and at least one hydrophilic area defined between the hydrophobiclines. A monolayer of assay beads can be provided at each of the atleast one hydrophilic areas. In some embodiments, the ordered array ofbeads comprises hydrophobic lines, a hydrophilic area, a combinationthereof, or multiples thereof, or a coated or uncoated surface, thatcomprises a pattern of photo-resist, polydimethylsiloxane, metal, glass,metal oxide, or plastic.

Various embodiments of a method is provided which includes washing anarray of first beads on a substrate, with a wash solution to removestacked beads from the substrate, the wash solution comprising from 2%to 50% by weight inert solid beads in a carrier, the first beads havingan average diameter and the solid beads having an average diameter thatis at least twice the diameter of the first beads, the washing formingdislodged first beads. In some embodiments, the methods include removingthe wash solution and the dislodged first beads from the substrate toform a monolayer of first beads. In some embodiments, the substrate caninclude a slide, and the substrate can also include a plurality ofchannels formed in a surface thereof.

In some embodiments, the first beads can include DNA-coated beads. Insome embodiments, the carrier can include water. In some embodiments,the wash solution can include first abrasive beads having a secondaverage diameter, and second abrasive beads having a third averagediameter, and the second average diameter is at least 50% greater thanthe third average diameter.

Various embodiments of a method of forming an ordered array of beads arealso disclosed herein. In some embodiments, the method includescontacting a plurality of first beads with a poly(ethylene glycol) (PEG)solution comprising a PEG having a molecular weight of about 350 Da orless, to form a bead mixture, and depositing the bead mixture on asurface of an array substrate. The PEG can include various molecularweights, for example, about 200 Da. In some embodiments, the arraysubstrate can include a slide having a plurality of grooves in a surfacethereof, and the depositing comprises depositing the bead mixture intothe grooves.

Various embodiments of an ordered array of beads are also providedherein. In some embodiments, the array of beads includes a substratecomprising hydrophobic lines in the shape of a grid and at least onehydrophilic areas defined between the hydrophobic lines. The array canalso include a monolayer of assay beads each of the at least onehydrophilic areas. In some embodiments, the ordered array of beads caninclude at least one of the hydrophobic lines and the at least onehydrophilic area comprise a pattern of photo-resist,polydimethylsiloxane, metal, glass, metal oxide, or plastic.

Various embodiments of a method of imaging a plurality ofuniformly-sized beads in a bead array are also provided herein. In someembodiments, the methods include arranging a plurality ofuniformly-sized beads in a plurality of grooves in a grooved substrateto form an array, the grooves being spaced apart by a pitch equal toabout 110% or more of a diameter of the beads, the beads forming aplurality of single-file rows of beads in the respective grooves,wherein adjacent beads in each respective row are touching one another,each bead comprises a respective analyte fixed to a surface thereof, andthe respective analyte of each bead is different than the respectiveanalyte of at least one other bead of the plurality. The method can alsoinclude exciting the plurality of beads in the array such that a portionof the beads become excited, emitting an increased radiation intensityrelative to a portion of the other beads, and imaging the area with anoptics system configured to resolve adjacent grooves of the plurality ofgrooves but not to resolve adjacent excited beads within a same groove,the imaging forming an image array data set. The method can also includeidentifying the position of each bead in the array and whether or not itis excited based on the array data set, wherein the positions ofunresolvable adjacent excited beads within a same groove are determinedfrom identifying unresolved groups of quantized length.

In some embodiments, the portion of the plurality of beads comprisesabout 30% or less of the total number of beads. In some embodiments, theexciting comprises exposing the array to at least four differentexcitation conditions, and the imaging comprises generating a pluralityof array data sets, each array data set representing the array under adifferent excitation condition. The method can further include furthercomprising determining a sequence of each different analyte based on theplurality of array data sets. In some embodiments, the pitch can be fromabout 125% to about 300% of the diameter, about 140% to about 160% ofthe diameter, etc.

Various embodiments of a system are also provided herein. In someembodiments, the system includes a substrate comprising a plurality ofgrooves formed in a surface thereof, the grooves being spaced apart fromone another at a pitch, and a plurality of beads disposed in the groovesand forming an array, wherein for each groove a plurality of the beadsforms a row of beads in the groove and adjacent beads in the row touchone another, the beads having a diameter, and the pitch being from about125% to about 300% of the diameter. In some embodiments, the system caninclude an imaging system having a resolution sufficient to resolveadjacent rows but insufficient to resolve adjacent beads within a samerow under excited conditions, the excited conditions comprisingconditions whereby increased radiation is emitted from the bead comparedto radiation emitted from the same bead under non-excited conditions,the imaging system being configured to generate an array data set. Thesystem can also include a processor configured to identify the positionof each bead in the array based on the array data set, wherein thepositions of unresolvable adjacent excited beads within a same grooveare determined from identifying unresolved groups of quantized length.In some embodiments, the system of claim 18, the pitch can be, forexample, from about 140% to about 160% of the diameter. In someembodiments, the system can further include an excitation sourceconfigured to direct an excitation beam toward the array under at leastone set of excitation conditions. In some embodiments, the at least oneset of excitation conditions comprises at least four different sets ofexcitation conditions.

Various embodiments of a device are also provided herein. In someembodiments, the device includes a substrate, and a plurality of postsextending from a surface of the substrate, wherein the plurality ofposts comprise a plurality of post clusters. In some embodiments, one ormore of the post clusters define bead receiving areas between posts,each bead receiving area is configured to retain a single bead of apredetermined bead diameter of about 10 μm or less, each post comprisesa base portion and a tip, and the cross-sectional area of the postdecreases in a direction from the base portion to the tip.

In some embodiments, the post clusters are arranged to provide acenter-to-center spacing of adjacent beads when disposed in the device,of from about 1.1 to about 1.9 times the diameter of a bead of thepredetermined diameter. In some embodiments, the post clusters arearranged to provide a center-to-center spacing of adjacent beads whendisposed in the device, of from about 1.2 to about 1.6 times thediameter of a bead of the predetermined diameter. In some embodiments,the post clusters are arranged to provide a center-to-center spacing ofadjacent beads, when disposed in the device, of about 1.5 times thediameter of a bead of the predetermined diameter. In some embodiments,the substrate comprises an injection-molded cyclo-olefin polymermaterial. In some embodiments, the device further includes a pluralityof beads including one bead at each bead receiving area.

In some embodiments, the device further includes at least one fiducialpost disposed in at least one of the bead receiving areas, the at leastone fiducial post configured to prevent a bead from being received inthe respective bead receiving area. The fiducial post can extend furtherfrom the surface than each post of the plurality of posts. In someembodiments, the device can further include a plurality of beadsdisposed in the bead receiving areas and forming an array, wherein thecenter-to-center spacing of the beads in the array is from about 115% toabout 190% of the diameter of the beads.

Various embodiments of a system comprising some embodiment of thepresently disclosed device is also provided herein. In some embodiments,the system further includes an imaging system having a resolutionsufficient to resolve adjacent rows but insufficient to resolve adjacentbeads within a same row under excited conditions, the excited conditionscomprising conditions whereby increased radiation is emitted from thebead compared to radiation emitted from the same bead under non-excitedconditions, and the imaging system being configured to generate an arraydata set. In some embodiments, the system further comprises a processorconfigured to identify the position of each bead in the array based onthe array data set.

Various embodiments of a method of loading a plurality of magnetic beadsinto grooves of a grooved plate are also provided herein. In someembodiments, the method includes arranging a plate comprising grooves ona support such that grooves are vertically arranged, the supportcomprising an inclined surface and the grooves comprising respectivefirst ends arranged adjacent the inclined surface, and respective opentops, and disposing a plurality of beads on the inclined surface suchthat the beads traverse the inclined surface and enter the grooves atthe first ends. In some embodiments, the method can also include guidingthe beads into the grooves in a manner such that beads in each grooveare aligned with one another in the respective groove, the guidingcomprising placing a guide wall adjacent the open tops in sufficientlyclose proximity to prevent the beads in any one of the grooves frommoving into another one of the grooves, and fixing the beads in thegrooves.

In some embodiments, the method further includes separating the guidewall from adjacent the open tops and separating the plate from thesupport. In some embodiments, the plate comprises a first portion and asecond portion, and the method comprises separating the first portionfrom the second portion after fixing the beads in the grooves andseparating the plate from the support.

In some embodiments, the fixing the beads in the grooves step caninclude comprises contacting the beads with a chemical fixing agent. Insome embodiments, fixing the beads in the grooves can includemagnetically attracting the beads against surfaces of the grooves orphotoactively attaching the beads to the surfaces of the grooves. Insome embodiments, guiding the beads into the grooves can includemagnetically attracting the beads into the grooves.

Various embodiments of a method of loading a plurality of magnetic beadsinto grooves of a grooved plate are also provided herein. In someembodiments, the method includes providing a plate. In some embodiments,the plate can include a load portion, an excess portion, a plurality ofgrooves each having a respective open first end on the excess portion, arespective closed second end on the load portion, and a respective opentop, the second ends being closed by a sidewall, the sidewall comprisingan interior surface facing the respective groove and an exterior surfacefacing away from the groove. The method can also include positioning amagnet adjacent the exterior surface of the sidewall, loading magneticbeads into the open ends of the grooves, magnetically attracting themagnetic beads into the grooves, using the magnet, such that the beadsin each groove align with one another, and separating the load portionfrom the excess portion. The method can also include fixing the beads inthe grooves.

These and other embodiments of the present disclosure are detailedbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure will be more fully understood from the followingdetailed description taken in conjunction with the accompanyingdrawings, in which:

FIGS. 1A-1D provide an overview of a microabrasive solution treatmentmethod according to various embodiments of the present teachings;

FIG. 2 is a white light image of a bead array that includes clumpedstacks of bead, taken from an Olympus microscope at 20× magnificationand then digitally enhanced to 400%;

FIG. 3 is a white light image of the same bead array substrate shown inFIG. 2 following a wash treatment according to various embodiments ofthe present teachings and enhanced to 600%;

FIG. 4 shows a deposition method by settling, without centrifugation in12% PEG 100 mM NaCl solution;

FIG. 5 shows bead deposition where the beads were deposited using asolution of 100 mM NaCl without a PEG additive;

FIG. 6 shows about 280,000 beads per panel deposited onto an orderedarray slide using as an additive a 200 Da MW PEG in 100 mM of NaCl;

FIG. 7 shows about 280,000 beads per panel deposited onto an orderedarray slide using as an additive a 350 Da MW PEG in 100 mM NaCl;

FIG. 8 shows a zoomed into image by way of pixel enhancement of the 20×optical image shown in FIG. 7 and showing 1-micron diameter individualbeads deposited into the grooves on an ordered array slide;

FIG. 9 shows the spacing of an ordered array deposition using a PEGadditive;

FIG. 10 shows the spacing of an ordered array deposition without using aPEG additive;

FIG. 11 shows the chemical structure of an exemplary PEG additive thatcan be used according to various embodiments of the present teachings;

FIG. 12 shows beads randomly distributed on a slide and having noparticular ordering;

FIG. 13 shows an embodiment according to the present teachings whereinbeads are arrayed in rows in V-grooves of a grooved plate;

FIG. 14 is a side view of a slide showing exemplary dimensions that canbe used for the groove angles and pitch in a configuration for orderingbeads having a diameter of 1 μm, according to various embodiments of thepresent teachings;

FIG. 15 shows a top plan view of the slide, grooves, and beads packedinto rows, shown in FIG. 14;

FIG. 16 is a simulated fluorescent image of the beads shown in FIGS.14-15, showing all of the beads fluorescing, and demonstrates resolvedrows of beads and unresolved beads within each of the rows;

FIG. 17 is a simulated fluorescent image of the beads shown in FIGS.14-15, showing a random 25% of the beads on the slide fluorescing,according to various embodiments of the present teachings;

FIG. 18 is a top view of a slide and bead array according to variousembodiments of the present teachings, wherein a slide is provided withstrategically placed injection molded posts useful to locate beadshaving a diameter of one micrometer (1 μm) in a hexagonal array;

FIG. 19 is a cross-sectional side view taken along line 19-19 of FIG.18;

FIG. 20 is a top view of a slide comprising posts according to variousembodiments of the present teachings, wherein the posts include afiducial post;

FIGS. 21A-21C depict three successive steps of a method andconfiguration for magnetically loading and fixing beads into grooves ofa slide, according to various embodiments of the present teachings; and

FIGS. 22A-22D depict four successive steps of a method and configurationfor magnetically loading and fixing beads into grooves of a slide, andremoving an excess slide portion, according to various embodiments ofthe present teachings.

DETAILED DESCRIPTION

According to various embodiments, a method is provided that compriseswashing an array of first solid-supports (e.g., beads) on a substrate,with a wash solution to remove stacked beads from the substrate. Thewash solution can comprise from about 2% by weight to about 50% byweight inert solid beads in a carrier, for example, from about 5% byweight to about 20% by weight, or from about 9% by weight to about 10%by weight. The first beads have an average diameter and the solid beadsin the wash solution have an average diameter that is larger than theaverage diameter of the first beads, for example, at least twice thediameter of the first beads. The washing can form dislodged first beads.The method can comprise removing the wash solution and the dislodgedfirst beads from the substrate to form a monolayer of first beads. Insome embodiments, multiple washes are used. In some embodiments, thesubstrate can comprise a slide. In some embodiments, the substrate cancomprise a plurality of channels formed in a surface thereof. In someembodiments, the first beads can comprise polynucleotide-coated beads orbeads coated (or at least partially coated with some other biomolecule).In some embodiments, the carrier can comprise water, alcohol, or anothercarrier that does not interact with polynucleotide-coated first beads.In some embodiments, the wash solution can comprise first abrasive beadshaving a second average diameter, and second abrasive beads having athird average diameter, and the second average diameter can be at least25% greater, at least 50% greater, or at least 75% greater than thethird average diameter.

According to various embodiments, the micro-abrasive bead wash solutionand method using the same removes stacked beads on ordered array slides,increasing usable beads for sequencing. The ability to remove stackedbeads increases the ability to deposit more beads per ordered arrayslide, which can increase throughput of a sequencing instrument. Theability to remove stacked beads results in a monolayer of beads on thesurface and aids in image focusing. Removing stacked beads enables thedetection of beads underneath a stack, whereas if there is a stack oftwo or more beads neither can be successfully interrogated. The abilityto remove stacked beads also reduces signal noise, which reduces errors.The ability to remove stacked beads also improves the functionality ofautomated imaging systems to detect beads. Moreover, the ability toremove stacked beads enables an increase in the number of beads perordered array slide, reducing the cost per sequenced base on asequencing instrument.

According to various embodiments, beads can be dispersed on a substrateto form a bead array. Initially, the beads can be dispersed in a randomfashion on a substrate, for example, on a glass slide. The initialdeposition can result in variation in bead density and, sometimes,clumping of beads. According to the present teachings, ordered arraysubstrates are provided to organize beads on the surface of a substratefor sequencing. To minimize bead stacking in high density arrays, amethod is provided to remove the stacked beads, reduce the production ofstacked beads, and reduce bead to bead interaction.

In some embodiments, a micro-abrasive bead wash solution and wash methodis provided to remove stacked beads from a slide following initial beaddeposition on the slide. After depositing a high density of beads, forexample, greater than about 200,000 beads per panel concentration, thedeposition chamber liquid can be exchanged with a micron bead solution.In some embodiments, the micron bead solution can act as an abrasive andremove most stacked beads on the slide, leaving a single layer of beads.By removing stacked beads, this micro-abrasive bead wash solution andthe method of using the solution enable increased density loading ofbeads. The result is increased instrument throughput as the array ofbeads thus formed on the surface can then be individually interrogated,for example, individually identified and sequenced.

According to various embodiments, a solution of beads is used as anabrasive to remove stacked beads on an ordered array slide. In someembodiments, following deposition of polynucleotide-coated beads onto anordered array slide substrate, a micro-abrasive bead solution can bepipette onto the slide, for example, inside a chamber covering thepolynucleotide-coated bead array. The solution can be caused to flowover the deposited beads. The beads in the micro-abrasive solution canbe made to bump into stacked beads on the slide, and disrupt theirattachment to other beads on the surface. This action removes thestacked beads, resulting in a monolayer of beads on the array slidesurface. The monolayer of beads can be interrogated, the beads can beindividually resolved, and the array can be useful for a ligation assay,a hybridization assay, or the like. The ability to clear away stackedbeads on an ordered array slide allows for increased deposition of beadsbeyond the saturation point that would otherwise be limiting due tostacked beads.

In some embodiments, the micro-abrasive bead can be monodispersed, thatis, of uniform-sized beads with little to no deviation in size. In someembodiments, the solution can comprise a mixture of two different sizedbeads, for example, comprising beads of about 3 micron average diameterand beads of about 5 micron average bead diameter. Larger beads can beused to facilitate movement of smaller beads, and in the example justdescribed, the 5 micron average diameter beads can facilitate movementof the 3 micron average diameter beads when they bang into each other.Depending on the size of the polynucleotide-coated beads forming themonolayer array, the larger sized beads can be used to disrupt largergroups of stacked beads.

The abrasive solution beads can comprise silica, silicon nitride, boronnitride, other inert, hard materials, or the like. The density of theabrasive solution beads can be from about 1.0 grams per cubic centimeter(g/cc) to about 3.0 g/cc, for example, from about 1.5 g/cc to about 2.5g/cc, or about 2.0 g/cc. In some embodiments, silica beads of a densityof about 2.0 g/cc are used.

FIGS. 1A-1D show an overview of a microabrasive method according tovarious embodiments of the present teachings. In FIG. 1A, an orderedarray slide 30 having a plurality of DNA-coated beads 32 is shown asDNA-coated beads 32 are initially deposited. In FIG. 1B, a solutioncontaining 3-5 micron abrasive beads 34 is poured over DNA-coated beads32 on the grooved surface 36 of slide 30. Slide 30, covered withmicro-abrasive solution and beads 34, is then is placed on a Roto-Torqueand rocked so that beads 34 in the micro-abrasive solution bombardstacked beads 38 and dislodge stacked beads 38 from surface 36, as shownin FIG. 1C. As can be seen in FIG. 1D, after treatment, themicroabrasive solution and dislodged beads are removed from surface 36and slide 30 is ready for sequencing.

FIG. 2 is a white light image taken from an Olympus microscope at 20×magnification and then digitally enhanced to 400%. The image is from anordered array slide after deposition of beads at a concentration of240,000 1024-synthetic beads per panel area. The image shows individualbeads, and clumped stacks 40 of beads. The circles mark a few examplesof clumped stacks 40 covering an underlying mono-layer of beads.

FIG. 3 is a white light image of a bead array taken with an Olympusmicroscope at 20× magnification. The image has been digitally enhancedto a 600% image. The image is of the same as that taken of FIG. 2 butafter the ordered array slide has been subject to a bead abrasive washtreatment according to the present invention. As can be seen, the imageshows that the wash greatly reduced the population of stacked beads inthe array, and increased the mono-layer area of the array asparticularly shown in the circled portions.

According to yet other various embodiments of the present teachings, toincrease the mappable bead loading on a slide, an ordered array slide isprovided that can align deposited beads into slots or grooves. Byaligning the beads on the surface, better bead-finding algorithms can bedesigned to detect and analyze the beads. In some embodiments, beads aredeposited on, for example, a metal-treated glass slide by settling onthe surface. Sometimes, however, random bead attachment all over theslide results, not just in the slots or grooves. Addition of the beadsto the deposition chamber, followed by immediate centrifugation, canresult in the beads depositing into the slot/grooves. In someembodiments, to increase bead binding within slots or grooves, a methodis provided to slow down bead attachment to the slide surface, whichallows the beads time to move into the slots or grooves beforeattachment to the surface. The method can utilize a novel additive tothe deposition solution to deposit the beads into the slots or grooves,without hindering the attachment chemistry.

According to various embodiments, a method of forming an ordered arrayof beads is provided that comprises contacting a plurality of firstbeads with a PEG solution comprising a PEG having an average molecularweight of about 350 Da or less, to form a bead mixture, and depositingthe bead mixture on a surface of an array substrate. In someembodiments, the PEG can have an average molecular weight of about 200Da. The array substrate can comprise a slide having a plurality ofgrooves formed in a surface thereof, and the bead mixture can bedeposited into the grooves.

In some embodiments, PEG is added to the bead deposition solution actsto slow down the attachment process and reduce bead-to-bead clumping andsticking, while still maintaining the bead attachment chemistry. Thisenables the use of an ordered array for SOLiD sequencing. Thedistribution of beads during bead deposition to form an ordered arraycan be dependent on the molecular weight of the PEG additive used in thedeposition solution. In some embodiments, the PEG used has a molecularweight range that aids bead deposition into the slots or grooves of theslide. In some embodiments, the PEG used has an average molecular weightof from about 100 Daltons (Da) to about 300 Da, for example, from about150 Da to about 250 Da, or about 200 Da. In some embodiments, not onlydoes the PEG additive aid in depositing the beads into the slidegrooves, but also results in a small, reproducible spacing between thebeads on the surface. This reduces the bead-to-bead contact. The resultis an increase in the ability of the bead mapping software to identifyand interpret bead reporter signals during sequencing.

FIG. 4 shows a deposition method by settling, without centrifugation in12% PEG 100 mM NaCl solution. The image shows high degree of beadstacking and clumping randomly all over the ordered array slide and notdeposited into the slots/grooves. The image is a white light image froman Olympus microscope at 20× magnification digitally enhanced to a 400%image.

FIG. 5 shows bead deposition after treatment with a solution of 100 mMNaCl without a PEG additive. In the array shown in FIG. 5, sonicatedbeads were delivered to a deposition chamber, the chamber was sealed,and then centrifuged within 2-minutes. The image shows improveddeposition into the grooves but also a high degree of bead stacking andclumping on the slide surface. The image is a white light image from anOlympus microscope at 20× magnification digitally enhanced to a 400%image.

FIG. 6 shows 280,000 beads per panel deposited onto an ordered arrayslide using as an additive a 200 Da MW PEG in 100 mM of NaCl. The imageis a white light image from an Olympus microscope at 20× magnificationdigitally enhanced to a 400% image.

FIG. 7 shows 280,000 beads per panel deposited onto an ordered arrayslide using as an additive a 350 Da MW PEG in 100 mM NaCl. The image isa white light image from an Olympus microscope at 20× magnificationdigitally enhanced to a 400% image.

FIG. 8 shows a zoomed into image by way of pixel enhancement of the 20×optical image shown in FIG. 7 and showing 1-micron diameter individualbeads deposited into the grooves on an ordered array slide. As can beseen, individual beads are clearly identifiable.

FIG. 9 shows the spacing of an ordered array deposition using a PEGadditive. The image represents beads to scale and single bead spreadfunction to determine distance. There is an almost regular gap betweenbeads with 23 beads shown in a 33.5 μm region showing an average spacingdistance of from about 0.1 to 0.15 microns.

FIG. 10 shows the spacing of an ordered array deposition without using aPEG additive. The image scanned represents beads to scale and usedsingle bead spread function to determine distance. As is shown, 24 beadsare distributed in the 33.5 μm region scanned. Essentially the same beaddeposition level as the regularly spaced beads results although in thiscase beads are clustered in groups of touching beads.

FIG. 11 shows the chemical structure of an exemplary PEG additive thatcan be used according to various embodiments of the present teachings,for example, that can be used in making an ordered bead array for aSOLiD system.

In some embodiments, the bead spacing or gap can be regular enough to bemeasured when compared to a non-PEG deposited ordered array slide. FIGS.5 and 6 show a comparison between an ordered array slide made accordingto the present teachings (FIG. 6) and an ordered array slide madewithout using PEG (FIG. 5). As can be seen, the PEG additive increasesthe interrogation ability of high density arrays and increases beadloading as well as keeps the beads isolated on the slide.

In some embodiments, a PEG molecule is added to the bead depositionsolution for forming a SOLiD (Life Technologies, Carlsbad, Calif.)ordered array. The PEG additive slows the attachment of DNA coated beadto the surface and results in evenly spaced beads on the surface. Insome embodiments, poly(ethylene glycol) having an average molecularweight of less than 350 Da was used, as shown in Example 2 below.Ethylene glycol, 2-methoxyethanol, 1,2-dimethoxyethanol poly(ethyleneglycol), a methoxy or dimethoxy substituted PEG molecule, a discrete PEGsuch as diethylene glycol or tetra(ethylene glycol), a mixture thereof,or the like, can be used, as exemplified in FIG. 11.

According to various embodiments, the method enables the ability todeposit high densities of beads into grooves of an ordered array slideand to deposit high densities of beads with reduced bead sticking,clumping, and stacking. The method enables the ability to deposit higherdensities of beads that are able to be individual mapped andreporter-detected, which increases instrument throughput per run. Themethod enables the ability to deposit beads side-by-side with a spacinggap between beads, which improves detection and isolation of each beadreporter. The method enables the ability to deposit beads side-by-sidewith a spacing gap between beads, which improves mapping of individualbeads, and enables the ability to deposit beads side-by-side with aspacing gap between beads, which reduces noise contribution reportedfrom adjacent beads. The method enables the ability to deposit beadswith reduced bead stacking, enabling better image focusing. Moreover,the method enables the ability to deposit beads in an ordered arrayslide, and allows for improved algorithms to detect and analyze eachindividual bead signal reporter. Furthermore, the method enables theability to deposit a higher concentration of beads on a surfaceefficiently and effectively, which reduces bead waste per deposition.

According to various embodiments, an ordered array of beads is providedthat comprises a substrate comprising hydrophobic lines in the shape ofa grid and at least one hydrophilic area defined between the hydrophobiclines. A monolayer of assay beads can be provided at each of the atleast one hydrophilic areas. In some embodiments, the ordered array ofbeads comprises hydrophobic lines, a hydrophilic area, a combinationthereof, or multiples thereof, that comprise a pattern of photo-resist,polydimethylsiloxane, metal, glass, metal oxide, or plastic.

According to yet other various embodiments of the present teachings,techniques for creating high capacity ordered arrays of beads areprovided. To accommodate a higher number of samples, a gasketarrangement can be used that comprises polydimethylsiloxane (PDMS) or asimilar material, to form separate confinement areas.

In some embodiments, hydrophobic separators are provided betweenhydrophilic sample confinement areas. For example, hydrophobic lines canbe used to form a grid with hydrophilic areas between. The hydrophobiclines can have a larger grid with wider hydrophobic spaces toaccommodate larger sample volumes, and the sample can cover severalhydrophilic areas. Yet larger lines creating larger areas, for example,from mid-sized areas composed of multiple smaller areas, can be created.

According to various embodiments, photo-resist, PDMS, or other similarmaterials can be used in place of a surface treatment. Plasma treatingover a mask can be used to change surface energy, and thus makedifferent regions hydrophilic or hydrophobic. Stamping ornano-imprinting, and silk screening, are additional options. In yetother embodiments, electro-wetting or opto-electro-wetting is used toform the appropriate patterns. In some embodiments, a surface treatmentis used to create hydrophobic and hydrophilic areas. Patterns ofphoto-resist, PDMS, metals, or other materials can also be made. Theresulting surface can then be further modified, if desired, to changethe surface energy. In some embodiments, the surface energy can bemodified by direct photo-modification. The photo-modification caninclude photo-cleavage, photo-activation, a combination thereof, and thelike. Patterning of light can be done using masks, DLP technology, LCDtechnology, a combination thereof, interference patterns, or the like.In some embodiments, devices, systems, and methods as described in thefollowing U.S. patents and Published Patent Applications can be used:U.S. Pat. No. 3,736,042; U.S. Pat. No. 4,705,705; U.S. Pat. No.5,143,854; U.S. Pat. No. 5,202,227; U.S. Pat. No. 5,324,591; U.S. Pat.No. 5,776,748; U.S. Pat. No. 5,985,551; U.S. Pat. No. 6,548,263; U.S.Pat. No. 6,921,636; US Patent Application Publication No. 2006/0286548;U.S. Pat. No. 4,447,140; U.S. Pat. No. 6,967,074; and U.S. Pat. No.5,510,270; each of which is incorporated herein in its entirety, byreference.

According to various embodiments, a clonal library confinement isprovided. As an alternative to a bridge amplification technique used tocreate randomly placed and spaced clonal clusters as a result of placingthe universal primers uniformly over the slide, a pattern of primers canbe used such that a grid is created. Such a pattern can be made usingmany of the methods described herein and described in the referencesincorporated herein. The methods can include using physical maskingtechniques by placing metal patterns to which desirable attachmentchemistry binds, or by placing metal patterns to which an undesiredchemistry does not bind. In some embodiments, the attachment of linkersto the primers can comprise reactions of photo-cleavable linkers, or thelinkers can be photo-activatable. In some embodiments, the primers canhave photo-cleavable terminators.

In yet other embodiments of the present teachings, physical barriers arecreated, such as micro-wells, using different materials which are notsuitable for an attachment chemistry of choice.

In some embodiments, single molecule sequencing is provided that useslight focusing devices, for example, light pipes or the like. Othermethods can be used to create a grid of attachment sites for targetnucleic acids.

To overcome a Poisson distribution that causes some areas to haveseveral different target molecules, and many with none, some embodimentsmake use of a zero mode waveguide technique, where a bead is attached tothe target. After attachment via covalent bonding hybridization,ligation, biotin-streptavidin, a combination thereof, or the like, thebead or similar object blocks any other bead from the area of interest.As a result, a high percentage of sites can be utilized. If desired, thebead can then be removed, or left in place, and can be selected so asnot to interfere with subsequent chemistry. Such an embodiment can beused with relatively larger or smaller features, for example, wells. Abead or similar target attachment structure can enable the isolation ofa single template molecule.

According to various embodiments, a bridge PCR system is providedembodying one or more of the bead manipulation embodiments describedherein. In some embodiments, a bead is used to physically blockadditional molecules from interaction with the target. A lowconcentration of beads, probably below that optimal for a Poissondistribution, can be used in some embodiments, for example, to perform aPCR reaction and/or another cyclical or repeatable reactions. An initialPCR reaction can be made to use most of the primers available in asingle area, preventing the possibility of amplification in the eventthat a second molecule happens to bind to a remaining unused primer.Thus, after several cycles, most of the available spaces can be filledwith clonal populations. In some embodiments, more cycles are used thanwould otherwise be used to ensure sufficiently complete usage ofprimers.

According to various embodiments, an ordered array maximizes thethroughput of a sequencing system, by maximizing the surface areautilization and minimizing the number of pixels needed per sequencingsite.

According to various embodiments, a method of imaging a plurality ofuniformly-sized beads in a bead array is provided. The method comprisesarranging a plurality of uniformly-sized beads in a plurality of groovesof a grooved substrate, to form an array. The grooves can be spacedapart from one another by a pitch equal to about 110% or more of adiameter of the beads. In some embodiments, the beads have a diameterand the grooves of the grooved plate are spaced apart from one anotherat a pitch of from about 125% to about 300% the diameter. In someembodiments, the pitch is from about 140% to about 160% of the diameter.

In some embodiments, the beads can be configured to form a plurality ofsingle-file rows of beads in the respective grooves. Adjacent beads ineach row can be touching one another, and each bead can comprise arespective analyte fixed to a surface of the bead. The respectiveanalyte of each bead can be different than the respective analyte of atleast one other bead of the plurality. The method can further compriseexciting the plurality of beads in the array such that a portion of thebeads become excited and emits increased radiation intensity relative toa portion of the other beads.

In some embodiments, the method can comprise imaging the array with anoptics system configured to resolve adjacent grooves of the plurality ofgrooves but not necessarily of sufficient resolution to resolve adjacentexcited beads within a same groove. The imaging can comprise forming animage array data set. The method can also comprise identifying theposition of each bead in the array. In some embodiments, whether or noteach bead is excited can be determined based on the array data set. Insome embodiments, the positions of unresolvable adjacent excited beadswithin a same groove can be determined from identifying unresolvedgroups of quantized length.

According to various embodiments, the portion of the plurality of beadsthat becomes excited can comprise about 30% or less of the total numberof beads. In some embodiments, the exciting can comprise exposing thearray to at least four different excitation conditions, and the imagingcan comprise generating a plurality of array data sets, with each arraydata set representing the array under a different respective one of theexcitation conditions. In some embodiments, the analyte can bepolynucleotides and the method can further comprise determining asequence of each different analyte based on the plurality of array datasets.

According to various embodiments, a system is provided that comprises asubstrate, a plurality of beads, an imaging system and a processor. Thesubstrate can comprise a plurality of grooves formed in a surfacethereof, and the grooves can be spaced apart from one another at apitch. The plurality of beads can be loaded in the grooves and form anarray. For each groove, a plurality of the beads can form a row of beadsin the groove and adjacent beads in the row can touch one another. Thebeads can be uniformly-sized of a given diameter, and the pitch can befrom about 125% to about 300% of the diameter. The imaging system canhave a resolution sufficient to resolve adjacent rows but insufficientto resolve adjacent beads within a same row under excited conditions.The excited conditions can be such that increased radiation is emittedfrom the bead compared to radiation emitted from the same bead undernon-excited conditions.

The imaging system can be configured to generate an array data set. Insome embodiments, the processor can be configured to identify theposition of each bead in the array based on the array data set. In someembodiments, the positions of unresolvable adjacent excited beads withina same groove are determined from identifying unresolved groups ofquantized length. If the quantized length is, for example, four units,it can be determined that there are four excited beads in a row formingthe quantized length. In some embodiments, the pitch is from about 140%to about 160% of the diameter. In some embodiments, the system furthercomprises an excitation source configured to direct an excitation beamtoward the array under at least one set of excitation conditions. Insome embodiments, the at least one set of excitation conditionscomprises at least four different sets of excitation conditions, forexample, corresponding to conditions to excite four differentfluorescent dyes useful for sequencing a polynucleotide.

In some embodiments, slides with V-grooves are used to order beads intoan array whereby individual beads of the array can be identified a largepacking densities can be used. For example, FIG. 12 shows beads atrandom distribution, less than full packing density, with unresolvablediffraction circles. According to various embodiments, a geometricallyordered separation is provided wherein a center-to-center separation ofbeads is present on the order of the diameter of the diffraction circle.Such an embodiment provides resolvable features at ordered coordinatesthat help recognition software identify bead locations. Ordering thebeads in both ‘x’ and ‘y’ dimensions with a separation approximating thediffraction circle provides resolution in both dimensions.

In some embodiments, a packing density of about a factor of two can beachieved by ordering beads in each respective row. Beads that are notseparated within the same row but instead touch each other can yielddifficult to resolve diffraction circles as shown in FIG. 16. In someembodiments, however, such a packing scheme is used in a process whereonly a sub-population of the collection of beads lights up in any givenimage. In an example, beads used in a DNA sequencing method comprise acollection of beads with four different sub-populations, specifically,one for each of the nucleic acid bases A, C, G, and T. In some suchapplications, only one base can generate a signal in any given image.The beads which light up will appear as single beads, or as a group of a“quantized” length, depending on how many beads there are in a group ofadjacent beads. An example of such a detection configuration is shown inFIG. 17, which makes clear gaps between groups. Such a configurationgreatly simplifies a bead location software algorithm. In eachsuccessive base image, the beads will light up in a different groupingpattern, which data can be used to add further redundancy to thelocation specifics or addresses to be used with the algorithm.

According to some embodiments, beads are ordered in a row in a V-shapedgroove, and the beads settle into the grooves. The rows can be separatedat a ‘pitch’ on the order of the diffraction circle. In someembodiments, the rows can be separated at a ‘pitch’ on the order ofabout 110% or more of the average diameter of the beads, or from about125% to about 300% the diameter, or from about 140% to about 160% of thediameter. The grooves can be created by etching, micromachining,nanoimprint lithography, and the like. In an exemplary embodiment, thebeads are about 1 μm in diameter, and the rows are imaged at 20×magnification such that the imaged pitch of the rows on a CCD detectorchip is equivalent to the length of five pixels.

According to various embodiments, photoactivatable attachment chemistrycan be used to fix the beads to surfaces of each V-shaped channel. Thisand other chemistries can be used the enable jostling and/or settling ofthe beads into tight conformity, for example, in a touching, single-fileorder in the groove, before actual attachment or fixing to the groovesurface.

FIG. 12 shows randomly distributed beads 100 on a slide 102, having noparticular ordering. The bead density is well below a maximum density.FIG. 12 also shows a plurality of unresolvable diffraction circles 104.FIG. 13, on the other hand, shows an embodiment according to the presentteachings wherein beads 102 are arrayed in rows 106 in V-grooves 108 ofa grooved plate 110. As can be seen in FIG. 13, grooved plate 110 cancomprise a glass slide 112 and a layer of nano-imprint material 114 on atop surface 116 of glass slide 112.

FIG. 14 shows exemplary dimensions, according to various embodiments,that can be used for a slide 118 and grooves 119 configured to orderbeads 120 having a diameter of 1 μm. Although any groove shape, size,and angle can be used, the angle of about 54.74° and a V-shaped grooveare exemplified because 54.74° is a silicon anisotropic etch angle. Inthe configuration shown in FIG. 14, each bead 120 contacts the surfacesof V-groove 119 at two contact points denoted 122 and 124. The 1.85 μmpitch is equivalent to the length imaged by five pixels of an exemplaryCCD detector when the image is magnified 20 times. In some embodiments,the pitch can be equivalent to the length of from about 3 to about 20pixels, from about 4 to about 10 pixels, or from about 5 to about 6pixels of a pixilated detector when the pitch is magnified 20 times.FIG. 15 is a top plan view of the beads packed into rows shown in FIG.14.

FIG. 16 shows a simulated fluorescent image of the beads shown in FIGS.14-15, with all of the beads fluorescing, and demonstrates resolved rowsand unresolved beads within rows. While the beads in each row areunresolved in FIG. 16, it is unlikely that all beads in a row wouldfluoresce at the same time in a DNA sequencing detection scheme. FIG. 17shows a simulated fluorescent image of the beads shown in FIGS. 14-15,but with a random 25% of the beads fluorescing. As can be seen in FIG.17, the beads appear either singly, or in unresolved groups of quantizedlength. According to various embodiments, even the unresolved groups ofquantized length can be used to identify beads. In FIG. 17, it isreadily discernable that two fluorescing beads can be identified with asingle non-fluorescing bead in between them, as shown by bead group 130.Similarly, two adjacent beads that are both fluorescing can beidentified at bead group 132.

By arranging beads as described herein, packing density can be maximizedwhile resolution requirements are satisfied for beads having a largediffraction circle relative to the actual bead size. In the examplesshown in FIGS. 14-15, beads can be ordered at two-fold the packingdensity of beads separated in both an ‘X’ and a ‘Y’ dimension, forexample, at two-fold the density of beads disposed in a two-dimensionalarray of wells. In some embodiments, 1 micrometer (1 μm) diameter beadscan be packed at a density of about 50 million beads per squarecentimeter (cm²) such that at a 20× magnification, 300,000 beads can beimaged in a single 4 megapixel image. The location algorithms used forrandomly packed beads break down at densities well below the densitiesthat can be resolved according to the present teachings. The presentmethod results in more robust bead location software algorithms andenables all of the beads on the slide to be readily addressable. TheV-shaped groove or channel design increases the surface area of theslide in contact with the bead which captures beads more securely thanbeads attached to a planar surface, which can be more exposed to flow.Moreover, the configurations shown in FIGS. 13-15 provide two attachmentpoints per bead rather than a single attachment point as is the casewhen attaching a bead to a planar surface.

According to various embodiments, the number of sequencable features perimage is provided to achieve high throughput. Geometric ordering of thefeatures results in higher feature density than random ordering, and insome embodiments hexagonal ordering is used to yield a maximumresolvable density. In some embodiments, the arrays comprise linearlyordered lmicron beads arranged in troughs. In some embodiments, PEG canbe used in the deposition buffer to retard the attachment process of thebeads to the trough walls until the beads are fully settled and centeredin the troughs. In some embodiments, PEG is used to impose a welldefined bead separation of around 0.2 microns between the 1 micronbeads. In some embodiments, such a spacing is used to facilitate beadidentification and reduce bead-to-bead cross talk. In some embodiments,the loss in bead density relative to a perfect touching hexagonalpacking, that results from keeping the beads slightly separated, is nota real loss in that without the separation many of the beads would notbe identifiable or would suffer cross-talk with neighboring beads,rendering them unuseful.

According to various embodiments of the present teachings, a device isprovided that comprises a substrate and a plurality of posts extendingfrom a surface of the substrate. The plurality of posts can comprise aplurality of post clusters wherein one or more of the post clustersdefine bead receiving areas between posts. Each bead receiving area canbe configured to retain a single bead of a predetermined bead diameter,for example, of a bead diameter of about 10 μm or less. In someembodiments, each post can comprise a base portion and a tip, and thecross-sectional area of the post can decrease in a direction from thebase portion to the tip. In some embodiments, the post clusters arearranged to provide a center-to-center spacing of adjacent beads whendisposed in the device, of from about 1.1 to about 1.9 times thediameter of a bead of the predetermined diameter. In some embodiments,the post clusters can be arranged to provide a center-to-center spacingof adjacent beads when disposed in the device, of from about 1.2 toabout 1.6 times the diameter of a bead of the predetermined diameter. Insome embodiments, the post clusters are arranged to provide acenter-to-center spacing of adjacent beads, when disposed in the device,of about 1.5 times the diameter of a bead of the predetermined diameter.In some embodiments, the substrate can comprise an injection-moldedpolymer, for example, an injection molded cyclo-olefin polymer material.

In use, the device can comprise a plurality of beads, including one beadat each bead receiving area. In some embodiments, at least one fiducialpost can be provided, disposed in at least one of the bead receivingareas. The at least one fiducial post can be configured to prevent abead from being received in the respective bead receiving area. Eachfiducial post can extend further from the surface than each post of theplurality of posts. In some embodiments, the device comprises aplurality of beads disposed in the bead receiving areas and forming anarray, wherein the center-to-center spacing of the beads in the array isfrom about 115% to about 200% of the diameter of the beads.

According to various embodiments, a system is provided that comprises adevice as described above, and an imaging system. The imaging system canhave a resolution sufficient to resolve adjacent beads in the array,under excited conditions. The excited conditions can comprise, forexample, conditions whereby increased radiation is emitted from the beadcompared to radiation emitted from the same bead under non-excitedconditions, and the imaging system being configured to generate an arraydata set. The system can further comprise a processor configured toidentify the position of each bead in the array based on the array dataset, and, for example, use a plurality of data sets to sequence anpolynucleotide analyte.

In some embodiments, the maximum density of usable beads is achieved bya hexagonal array but wherein the beads are separated by just thatamount of separation required to optimize bead identification andminimize bead-to-bead cross-talk. According to various embodiments, aninjection molded polymer slide is used. Materials for such slides canbe, for example, cyclo-olefin polymers. As exemplified in FIGS. 18 and19, a slide 140 is provided with strategically placed injection moldedposts 142 useful to locate beads 144 having a diameter of one micrometer(1 μm) in a hexagonal array 146. A separation distance between centersof 1.2 μm is provided between the centers of beads 144 by the locationsof posts 142. Posts 142 can comprise a widened base portion 148 thatnarrows in cross-section as it comes to a tip 150 as shown in FIG. 19.FIG. 19 is a cross-sectional view taken along line 19-19 of FIG. 18. Thetapered configuration of the posts facilitates ready entry of the beadsinto their desired locations, and locates the beads with a high degreeof precision due to widened base portion 148. The dimensions shown areexemplary only, and not intended to limit the present teachings. Asshown in FIG. 19, each post 142 has a rounded post top and intersectswith a top surface 141 of slide 140 at an angle of 70° in the exemplaryembodiment.

The formation of posts 142, while exemplified by injection molding, canbe made by other methods, for example, by photoresist on a glass orpolymer substrate, by micromaching, chemical etching, vapor deposition,and the like. In some embodiments, the maximum obtainable density ofbeads is obtained by ordering them into a hexagonal Array. Differentpost sizes, post shapes and, and post spacing can be used to controlseparation of beads of different diameters and/or to increase resolutionunder specific conditions. The posts can provide a separation thatcannot be achieved, for example, by a self-assembled-monolayer (SAM),while at the same time the posts maintain density optimization of ahexagonal packing.

According to various embodiments, posts are used on a slide to provideoptimal density packing of the beads on the slide and to provideentrapment of the beads. The entrapment can be sufficient to make themresistant to dislodgement by a fluid flow. Without entrapment or similarfixing of the beads, the result would be a loss of beads or wash-out ofbeads due to fluid flow over many cycles of flow.

According to various embodiments, a slide comprising a configuration ofposts is provided wherein the arrangement of the posts permits a freefluid exchange between each bead and the surrounding fluid. The benefitis pronounced compared to devices wherein a bead is located in a fullysurrounding well, particularly considering that the fit between the beadand the well would need to be fairly close in order to accurately locatethe bead. Moreover, the benefit is pronounced in situations whereinreactions and viewing occur on the underside of the bead, at leastbecause the underside of a bead in a fully surrounding well wouldexperience impeded fluid exchange.

FIG. 20 is a top view of a slide comprising posts according to variousembodiments of the present teachings, wherein the posts include afiducial post. As shown, a slide 160 is provided with an additional post162 provided at the center of a post cluster formed by posts 164, 165,166, 167, 168, and 169. In an example, additional post 162 can be, forexample, larger than the surrounding posts and/or of a configurationthat prevents a bead from being accepted or received at that location.Additional post 162 can exclude a bead from that position and/or act asa fiducial. In some embodiments, a pattern or array of additional posts162, or filler posts, can be employed, for example, to code differentimage panels with different fiducial codings. In the embodiment,exemplary dimensions include posts having a base portion that intersectswith a top surface of the slide at a circle having a diameter of 1.386μm.

In some embodiments, the slides can be used with the Life Technologies(Carlsbad, Calif.) SOLiD platform.

According to various embodiments, a method of loading a plurality ofmagnetic beads into grooves of a grooved plate is provided. The methodcomprises first arranging a plate comprising grooves on a support suchthat grooves are vertically arranged. The support comprises an inclinedsurface and the grooves comprise respective first ends arranged adjacentthe inclined surface, and respective open tops. A plurality of beads arethen disposed, poured, or otherwise placed on the inclined surface suchthat gravitational, magnetic, centrifugal, or other forces can be usedto cause the beads to traverse the inclined surface and enter thegrooves at the first ends. The beads can be guided into the grooves in amanner such that beads in each groove are aligned with one another ineach respective groove.

The guiding can comprise placing a guide wall adjacent the open tops insufficiently close proximity to prevent the beads in any one of thegrooves from moving into another one of the grooves. Subsequently, thebeads can be fixed in the grooves. In some embodiments, the methodfurther comprises separating the guide wall from adjacent the open topsand separating the plate from the support. The plate can comprise afirst portion and a second portion, and the method can also compriseseparating the first portion from the second portion after fixing thebeads in the grooves, for example, also after separating the plate fromthe support. The beads can be fixed in the grooves by contacting thebeads with a chemical fixing agent, magnetically attracting the beadsagainst surfaces of the grooves, combinations thereof, or the like. Insome embodiments, the beads can be guided into the grooves bymagnetically attracting the beads into the grooves.

According to some embodiments, sequencing beads are placed in grooves ofa grooved slide. The device and method can be used to increase packingdensity relative to a non-grooved slide by setting a small, uniform gapor pitch between rows of beads. The device enables beads to touch onlyadjacent beads along the dimension of the respective row defined by agroove of the slide. The device and method using same improve imageresolution and interpretation. To minimize gaps between beads within thesame row, a magnetic loading can be used according to variousembodiments, as illustrated in FIGS. 21A-21C.

FIGS. 21A-21C depict a loading system and three successive steps of aloading method for magnetically loading and fixing beads into grooves ofa slide. FIG. 21A shows a system 210 for loading a plurality of magneticbeads 212 into a slide 214 having a plurality of grooves 216. The systemincludes a support 211, and a slide 214. Support 211 comprises a firstguide wall 218 and a second guide wall 220 that together are configuredto guide beads 212 in a manner such that beads 212 slide into grooves216 of slide 214. Support 211 can also comprise sidewalls 222 and 224 tohelp guide beads 212 into grooves 216. Beads 212 are magnetically forcedinto stacking inside grooves 216 by a magnet, and in the embodimentshown, a magnet 226 is provided to magnetically attract beads 212 intogrooves 216. As magnet 226 pulls beads 212, guide walls 218 and 220direct beads 212 into grooves 216. In some embodiments, the pitch of thegrooves is from about 1.1 to about 2.5 times the bead diameter, forexample, from about 1.3 to about 1.9 times, from about 1.4 to about 1.6times, or about 1.5 times the bead diameter. For example, if beadshaving a diameter of 1.0 micrometer (μm) are to be used, the pitch ofthe grooves can be about 1.5 μm. After beads 212 are stacked in grooves216, the resulting filled grooves are as shown in FIG. 21B.

In a next step of the method, and as shown in FIG. 21B, a second magnet228 can be used to pull beads 212 against the bottom surface or surfacesof grooves 216. While a V-shaped cross-section groove is depicted, othergroove shapes can be used, for example, a U-shaped groove or the like.In addition, or as an alternative, a fixing chemistry, depicted asdroplet 230, can be used to chemically fix beads 212 in the grooves 216.While magnetic attraction and chemical treatments are exemplified asmethods of fixing the beads in the grooves, other fixing methods can beused. Subsequent to fixing beads 212 in grooves 216, slide 214 bearingbeads 212 can be separated from support 211 as an assembly 232.

FIG. 21C shows assembly 232 removed from support 211 and ready to loadinto an instrument, for example, into a DNA sequencing device. Anexemplary platform that can be used with assembly 232 is the SOLiDsequencing platform available from Life Technologies, Carlsbad, Calif.

According to yet another embodiment of the present teachings, a methodof loading a plurality of magnetic beads into grooves of a grooved plateis provided. The method can comprise use of a plate comprising a loadportion, an excess portion, a plurality of grooves each having arespective open first end on the excess portion, a respective closedsecond end on the load portion, and a respective open top. The secondends can be closed by a sidewall and the sidewall can comprise aninterior surface facing the grooves and an exterior surface facing awayfrom the grooves. A magnet can be positioned or provided adjacent theexterior surface of the sidewall. Magnetic beads can then be loaded intothe open ends of the grooves and can be magnetically attracted into thegrooves, using the magnet.

In some embodiments, the beads in each groove can align with oneanother, for example, in a single file manner. After bead loading, theload portion of the plate can be separated from the excess portion ofthe plate. In some embodiments, the method can further comprise fixingthe beads in the grooves, for example, with a chemical, with a magnet,with a combination thereof, or the like.

FIGS. 22A-22D depict four successive steps of a method and configurationfor magnetically loading and fixing beads into grooves of a slide, andremoving an excess slide portion, according to various embodiments ofthe present teachings. In FIG. 22A, a slide assembly 300 is providedinto which a plurality of beads 302 are loaded. Beads 302 are forcedinto the slide, for example, by gravity or centrifugal force, and landrandomly in grooves 304 formed in slide assembly 300. In someembodiments, the pitch of the grooves is from about 1.1 to about 1.9times the bead diameter, for example, from about 1.3 to about 1.7 times,from about 1.4 to about 1.6 times, or about 1.5 times the bead diameter.For example, if beads having a diameter of 1.0 micrometer (μm) are to beused, the pitch of the grooves can be about 1.5 μm.

As seen in FIG. 22B, a magnet 306 located at a side and slightly belowslide assembly 300, is used to attract beads 302 such that they stack upagainst a side wall 308 of slide assembly 300 and down into grooves 304.

FIG. 22C depicts a chemistry 310 being applied, and/or depicts theapplication of a new environmental condition, that fixes beads 302 intogrooves 304. As shown in FIG. 22D, an excess portion 312 of slideassembly 300 is removed by cutting, breaking, ungluing, frictionrelease, or the like, leaving a slide read assembly 314. Slide readassembly 314 is then ready to load into an instrument, for example, intoa DNA sequencing device. An exemplary platform that can be used withassembly slide read assembly 314 is the SOLiD sequencing platformavailable from Life Technologies, Carlsbad, Calif. The throughput ofsuch a device can be, for example, 60 GB for a run with 2 slides of 1 μmbeads, using a 1.5 μm pitch between rows, considering a 68% slide fillfactor, using DNA fragments of 35 base pairs (bp) on each bead.

Other devices, systems, and methods that can be used in conjunction withthe present teachings include those described in US Patent ApplicationPublications Nos. 2003/0165935 A1, published on Sep. 4, 2003, and2005/0118589 A1, published on Jun. 2, 2005, both of which areincorporated herein in their entireties by reference.

The present teachings will be more fully understood with reference tothe following Examples that are intended to illustrate, not limit, thepresent teachings.

EXAMPLES Example 1

DNA-coated beads DH10 fragment library control SOLiD PN 4392462, AppliedBiosystems, LLC, Foster City, Calif., are deposited at a loading densityof 240,000 Control beads per panel area, on a zirconium coated orderedarray slide. After pipetting the beads into a deposition chamber usingstandard SOLiD 3 deposition equipment and conditions, the slide, in thedeposition chamber assembly, was centrifuged for 5 minutes at a force of325×g=RCF, and the beads were bound within the groves of the orderedarray slide.

FIG. 2 is a digital image of the resulting array, captured using aMETAMORPH software package and Olympus microscope at 20× magnification.The image was digitally enhanced to 400% image after centrifugation. Thedark clumps shown in the image of FIG. 2 are stacked beads.

A micro-abrasive bead wash solution was prepared by mixing together, ina 1.5 mL Eppendorf tube: 500 μL of 9.8% solid, 3.01 micron diameterSilica beads, from Bangs Laboratories, part number-SS05N/5411; and 100μL of 10% solid, 4.74 micron diameter Silica beads, Bangs Laboratories,part number-SS05N/5568. The mixture was then centrifuged for two minutesin an Eppendorf centrifuge model 5415 at speed 14 (16,000×g=RCF). Theliquid above the bead pellet was pipetted off and replaced with an equalamount of 100 mM NaCl. The mixture was vortexed and then centrifuged fortwo minutes in an Eppendorf centrifuge model 5415 at speed 14. Theliquid above the bead pellet was pipetted off and raised to a finalvolume to 1000 μL of 100 mM NaCl.

The solution was then vortexed until the beads were completelyresuspended. After the DNA-coated beads were deposited on the surface,the bead deposition buffer was removed from the chamber and enoughabrasive bead solution was added to fill the volume of the chamber 60%.The partial filling left space in the chamber for the bead solution toflow within the chamber. The chamber with abrasive bead wash was placedonto a Roto-Torque rotation device with the rotation set to Low and therotation speed was set to 7. The rotary was tilted approximately 60° androtated for 15 minutes at room temperature. The rotation allowed theabrasive beads to roll over the deposited bead surface and to knock intothe stacked beads. The disruption left a monolayer of beads on thesurface. After 15 minutes, the abrasive bead solution was removed andthe slide chamber was rinsed to remove wash solution beads and thedisrupted DNA-coated beads.

The 240,000 bead deposition slide was then imaged again. FIG. 3 showsthe result of the abrasive bead solution treatment showing most of thestacked beads removed. The treatment increased the number ofindividually detectable beads on the surface for sequencing.

Example 2

DNA-coated beads (DH10 fragment library control SOLiD PN 4392462—AppliedBiosystems, LLC, Foster City, Calif.), with a loading of 260,000 Controlbeads per panel area, were deposited on a zirconium coated ordered arrayslide. The SOLiD DH10 Library control beads were prepared fordeposition, on the SOLiD ordered array slides, by the following protocolresulting in the monolayer demonstrated in FIG. 6.

Step 1—In a first step, an aliquot of beads was dispensed into a 1.5 mLEppendorf tube and placed into a magnet separator holder. After waitinga period of 2 minutes, the beads had collected on the side of the tubewith the magnet and the solution was removed.

Step 2—A solution of 100 mM NaCl (100 μL) was added to the tube with thebeads. After removing from the magnetic holder, the solution with beadswas mixed by brief vortexing and then spun briefly at low RCF force witha table top centrifuge to pellet the liquid in the tube. The tube wasthen placed into the magnetic separator holder. After waiting a periodof 2 minutes, the beads had collected on the side of the tube with themagnet and the solution was removed.

Step 3—Step 2 was repeated.

Step 4—A solution of 12% (V/V) PEG (200 MW) in 100 mM NaCl (100 μL) wasadded to the beads. The tube was then removed from the magnetic holder,mixed by brief vortexing and then spun briefly at low RCF force with atable top centrifuge to pellet the liquid in the tube. The tube was thenplaced into the magnetic separator holder, After waiting a period of 2minutes, the beads had collected on the side of the tube with the magnetand the solution was removed.

Step 5—More of the solution of 12% (V/V) PEG (200 MW) in 100 mM NaCl(500 μL) was added to the tube with beads. The tube was then removedfrom the magnetic holder, mixed by brief vortexing and then spun brieflyat low RCF force with a table top centrifuge to pellet the liquid in thetube.

Step 6—The tube containing the PEG mixture with the beads was thenplaced into a Covaris sonicator and sonicated using the Enrich 01protocol. After Covaris treatment, the tube was removed from the deviceand spun briefly at low RCF force with a table top centrifuge to pelletthe liquid in the tube.

Step 7—The solution of DNA coated beads in the PEG containing solutionwas then pipetted into a full slide SOLiD deposition chamber containinga zirconium coated ordered array slide. The chamber was tilted slightlyto allow the liquid to flow up in one continuous slow, even, bubble-freefront. After the chamber was full, sticky tabs were used to completelyseal the chamber holes.

Step 8—The deposition chamber was placed into Beckman AllegraCentrifuge, with a swinging bucket rotor, and spun at 162-197 RCF for 5minutes. FIGS. 6 and 8 show the beads on the slide following deposition.

In Example 2, the Polyethylene Glycol that was used had an average MW of200 DA, available as part number P3015 from Sigma Aldrich Chemical. TheNaCl that was used is available from Sigma as part number S-3014. The100 mM NaCl solution was prepared in deionized sterile water.

The Covaris Enrich01 protocol used was as follows:

Number of Cycles=4

Bath Temperature Limit (C)=20.000000

Mode=“Frequency Sweeping”

Water Quality Testing=FALSE

[Treatments]

Number of Treatments=4

[Treatment 0]

Cycles per Burst=1

Time(sec)=5.000000

Duty Cycle (%)=2.000000

Intensity=6.000000

[Treatment 1]

Cycles per Burst=1

Time(sec)=30.000000

Duty Cycle (%)=5.000000

Intensity=9.000000

[Treatment 2]

Cycles per Burst=0

Time(sec)=0.000000

Duty Cycle (%)=0.100000

Intensity=0.100000

[Treatment 3]

Cycles per Burst=0

Time(sec)=0.000000

Duty Cycle (%)=0.100000

Intensity=0.100000

One skilled in the art will appreciate further features and advantagesof the present disclosure based on the above-described embodiments.Accordingly, the present disclosure is not to be limited by what hasbeen particularly shown and described, except as indicated by theappended claims. All publications and references cited herein areexpressly incorporated herein by reference in their entirety.

What is claimed is:
 1. A method of preparing a sequencing apparatus, themethod comprising: applying a solution to a substrate including aplurality of microwells exposed at a surface of the substrate, aplurality of polynucleotide beads depositing at the surface of thesubstrate, a first portion of the plurality of polynucleotide beadsdepositing into microwells of the plurality of microwells, a secondportion of the plurality of polynucleotide beads depositing betweenmicrowells of the plurality of microwells, the solution comprising aplurality of inert beads to dislodge the second portion of thepolynucleotide beads from between the microwells; and removing thesolution including the plurality of inert beads and the second portionof the plurality of polynucleotide beads.
 2. The method of claim 1,wherein the solution includes about 2% by weight to about 50% by weightof the plurality of inert beads.
 3. The method of claim 2, wherein thesolution includes about 5% by weight to about 20% by weight.
 4. Themethod of claim 1, wherein an average diameter of the plurality of inertbeads is greater than an average diameter of the plurality ofpolynucleotide beads.
 5. The method of claim 1, wherein the averagediameter of the plurality of inert beads is at least twice the averagediameter of the plurality of polynucleotide beads.
 6. The method ofclaim 1, wherein an average diameter of the plurality of inert beads isin a range of 3 micrometers to 5 micrometers.
 7. The method of claim 1,wherein the plurality of inert beads includes a first set of inert beadshaving a first average diameter and a second set of inert beads having asecond average diameter at least 25% greater than the first averagediameter.
 8. The method of claim 7, wherein the second average diameteris at least 50% greater than the first average diameter.
 9. The methodof claim 8, wherein the second average diameter is at least 75% greaterthan the first average diameter.
 10. The method of claim 1, wherein theplurality of inert beads comprise silica, silicon nitride, or boronnitride.
 11. The method of claim 1, wherein the plurality of inert beadshave a density in a range of 1.0 g/cc to 3.0 g/cc.
 12. The method ofclaim 11, wherein the density is in a range of 1.5 g/cc to 2.5 g/cc. 13.The method of claim 1, wherein the plurality of microwells are spaced ata pitch of about 125% to 300% of an average diameter of the plurality ofpolynucleotide beads.
 14. The method of claim 13, wherein the pitch isabout 140% to 160% of the average diameter of the plurality ofpolynucleotide beads.
 15. The method of claim 1, further comprisingloading the substrate into a sequencing apparatus following removing thesolution.
 16. The method of claim 1, further comprising detectingdeposited polynucleotide beads following removing the solution.
 17. Themethod of claim 1, further comprising performing a sequencing processfollowing removing the solution.
 18. A method of preparing a sequencingapparatus, the method comprising: applying a solution to a substrateincluding a plurality of microwells exposed at a surface of thesubstrate, a plurality of polynucleotide beads depositing at the surfaceof the substrate, a first portion of the plurality of polynucleotidebeads uniquely depositing into microwells of the plurality ofmicrowells, a second portion of the plurality of polynucleotide beadsdepositing between microwells of the plurality of microwells, thesolution comprising a plurality of inert beads having an averagediameter at least twice the average diameter of the plurality ofpolynucleotide beads; and removing the solution including the pluralityof inert beads and the second portion of the plurality of polynucleotidebeads.
 19. The method of claim 18, further comprising loading thesubstrate into a sequencing apparatus following removing the solution.20. The method of claim 18, further comprising detecting depositedpolynucleotide beads following removing the solution.